ERYTHROPOIETIN--STRUCTURE FUNCTION RELATIONSHIPS

促红细胞生成素--结构功能关系

• 批准号: 3486233
• 负责人: H. Franklin Bunn
• 金额: $ 36.76万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3486233
• 关键词: Escherichia coli; Insecta; X ray crystallography; acid base balance; antigens; biological signal transduction; calcium flux; cell free system; chemical structure function; computer simulation; erythropoietin; genetic manipulation; glycosylation; heme; laboratory mouse; membrane permeability; nucleic acid biosynthesis; phenylhydrazines; protein biosynthesis; protein structure function; radiotracer; receptor binding; site directed mutagenesis; tissue /cell culture; transfection; tritium

One of the major unanswered questions in the regulation of hematopoiesis is the mechanism by which soluble growth factors interact with specific receptors on hematopoietic cells. This proposal focuses on establishing the structural features of erythropoietin (Epo) that are responsible for its biosynthetic processing and function. Site-directed mutations, both deletions and amino acid replacements, will be introduced into the Epo gene in order to test specific functional properties. Methods will be developed for high level production of normal and mutant Epo's in Cos7, CHO and S. frugiperda cells as well as in E. Coli and in a cell-free translation system. The recombinant Epo products will be purified to homogeneity and characterized biochemically. the following assays will be developed to assess both short-term and long-term effects of normal and mutant products in the Epo-responsive cell line HCD57: equilibrium binding, internalization, Ca2+ influx, intracellular pH, [3H]-thymidine incorporation, and heme synthesis. For the latter 3 measurements, HCD57 cells will be compared to erythroid cells isolated from the spleens of mice treated with phenylhydrazine. We will prepare cassette mutants that correspond to surface epitopes predicted from primary and secondary structure. The properties of these mutants will provide information on the domains of Epo responsible for binding to its receptor as well as for internalization. Particular epitopes of interest will be selected for preparation of additional mutants that will better define these functionally important sites. Site-directed mutants will also be used to investigate the biosynthetic and functional roles of post- translational modifications including N-terminal processing, cleavage of C-terminal Arg, as well as N- and O- linked glycosylation. This cumulative information will be used in developing a computer based prediction of 3-dimensional structure. Concurrently we will attempt to prepare crystals of normal Epo and selected mutants, and if successful, they will be analyzed by x-ray diffraction. From these studies we hope to gain comprehensive information on structure-function relationships of Epo that will contribute to an understanding, at the molecular level, of receptor binding, internalization and signal transduction.

造血调节中的主要未解决问题之一 是可溶性生长因子与特定相互作用的机制 造血细胞上的受体。 该提议着重于建立 促红细胞生成素（EPO）的结构特征负责 它的生物合成处理和功能。 位置定向的突变，两者均 缺失和氨基酸替代物将引入EPO 基因以测试特定的功能特性。 方法将是 开发用于高水平在COS7中的正常和突变EPO的生产， Cho和S. frugiperda细胞以及大肠杆菌和无细胞中 翻译系统。 重组EPO产品将被纯化为 同质性和生化特征。 以下测定将 开发以评估正常的短期和长期影响 EPO响应细胞系HCD57中的突变产物：平衡 结合，内在化，Ca2+流入，细胞内pH，[3H] - 胸腺胺 掺入和血红素合成。 对于后3个测量值，HCD57 将将细胞与从脾脏分离的红细胞细胞进行比较 用苯氢嗪处理的小鼠。 我们将准备盒式突变体 这对应于原发性和次级预测的表面表位 结构。 这些突变体的特性将提供有关的信息 EPO的领域负责与其受体结合以及 用于内在化。 将选择特定感兴趣的表位 为了准备其他可以更好地定义这些突变体 功能重要的站点。 现场定向的突变体也将使用 研究后的生物合成和功能作用 翻译修改包括N末端处理，裂解 C末端ARG以及N和O链接的糖基化。 这 累积信息将用于开发基于计算机的信息 3维结构的预测。同时我们将尝试 准备正常EPO和选定突变体的晶体，如果成功， 它们将通过X射线衍射进行分析。 从这些研究中我们希望 获取有关结构功能关系关系的全面信息 EPO将有助于理解分子水平的理解 受体结合，内在化和信号转导。